Atherosclerosis and vascular thrombosis are a major cause of morbidity and mortality, leading to coronary artery disease, myocardial infarction, and stroke. Atherosclerosis begins with an alteration in the endothelium which lines the blood vessels. An endothelial alteration may eventually result in the development of an endothelial lesion caused, in part, by the uptake of oxidized low-density lipoprotein (LDL) cholesterol. Rupture of this lesion can lead to thrombosis and occlusion of the blood vessel. In the case of a coronary artery, rupture of a complex lesion may precipitate a myocardial infarction, whereas in the case of a carotid artery, stroke may ensue.
In atherosclerotic coronary heart disease, endothelial dysfunction may diminish production of vasodilatory substances, such as nitric oxide. Myocardial ischemia results when autoregulatory vasodilation is prevented, whether by flow-limiting coronary arterial stenosis or by endothelial dysfunction. In both cases, arterial blood flow can no longer increase proportional to rising oxygen demands. In other situations, myocardial ischemia may occur when oxygen demands are constant but there is a primary decrease in coronary blood flow mediated via coronary artery spasm, rapid evolution of the underlying atherosclerotic plaque leading to a reduced coronary arterial lumen caliber, and/or intermittent microvascular plugging by platelet aggregates.
Balloon angioplasty is commonly used to reopen a blood vessel which is narrowed by plaque. Although balloon angioplasty is successful in a high percentage of the cases in opening the vessel, it often denudes the endothelium and injures the vessel in the process. This damage causes the migration and proliferation of vascular smooth muscle cells of the blood vessel into the area of injury to form a lesion, known as myointimal hyperplasia or restenosis. This new lesion leads to a recurrence of symptoms within three to six months after the angioplasty in a significant proportion of patients.
In atherosclerosis, thrombosis and restenosis there is also a loss of normal vascular function, such that vessels tend to constrict, rather than dilate. The excessive vasoconstriction of the vessel causes further narrowing of the vessel lumen, limiting blood flow. This can cause symptoms such as angina (if a heart artery is involved), or transient cerebral ischemia (i.e. a “small stroke”, if a brain vessel is involved). This abnormal vascular function (excessive vasoconstriction or inadequate vasodilation) occurs in other disease states as well. Hypertension (high blood pressure) is caused by excessive vasoconstriction, as well as thickening, of the vessel wall, particularly in the smaller vessels of the circulation. This process may affect the lung vessels as well causing pulmonary (lung) hypertension. Other disorders known to be associated with excessive vasoconstriction, or inadequate vasodilation include transplant atherosclerosis, congestive heart failure, toxemia of pregnancy, Raynaud's phenomenon, Prinzmetal's angina (coronary vasospasm), cerebral vasospasm, hemolytic-uremia and impotence.
A substance released by the endothelium, initially referred to as “endothelium derived relaxing factor” (EDRF), plays an important role in inhibiting these pathologic processes. EDRF is now known to be nitric oxide (NO). NO plays many roles in human physiology, including the relaxation of vascular smooth muscle, the inhibition of platelet aggregation, the inhibition of mitogenesis, the proliferation of vascular smooth muscle, and leukocyte adherence. Because NO is the most potent endogenous vasodilator, and because it is largely responsible for exercise-induced vasodilation in the conduit arteries, enhancement of NO synthesis could also improve exercise capacity in normal individuals and those with vascular disease.
Endothelial nitric oxide synthase (eNOS) is the nitric oxide synthase (NOS) isoform responsible for the maintenance of systemic blood pressure, vascular remodeling and angiogenesis (Shesely et al., 1996; Huang et al., 1995; Rudic et al., 1998; Murohara et al., 1998). As deficient endothelial production of NO is an early, persistent feature of atherosclerosis and vascular injury, eNOS has proven to be an attractive target for vascular gene therapy. While the regulation of eNOS activation remains largely undefined, it is known that eNOS is phosphorylated in response to various forms of cellular stimulation (Michel et al., 1993; Garcia-Cardena et al., 1996; Corson et al., 1996), however, the role of phosphorylation in the regulation of nitric oxide (NO) production and the kinase(s) responsible has not been previously elucidated.